CA1074699A - Treatment of viral infections - Google Patents

Abstract

TITLE
TREATMENT OF VIRAL INFECTIONS
INVENTOR
Vidya S. Gupta ABSTRACT OF THE DISCLOSURE
The compound 5-methoxymethyldeoxyuridine has been found to have useful therapeutic properties. Animal tests have shown antiviral activity against herpes simplex vi-ruses, e.g., in treatment of herpes keratitis and herpes encephalitis. An enhanced activity has been observed when administered in conjunction with arabinosyl adenine. Immu-nostimulant properties have also been observed in vivo.

Description

4ti~9 Field of -~he l`'.V~nti on Tn~s inverltion is concerned with the therapeutic use of 5-rnethoxymethyldeoxyuridine (1-(2-deoxy-~-D-ribo-furanosyl)-5-me-thoxymethyluracil), MMUdP~, in animals in-cluding man as an antiviral agent or as an immunostimu]ant or both. This compound MMUdR has shown very si.gnifi.cant activi-ty _ vivo against cer-tain herpes viruses, particu-larly herpes simplex (HSV-l). This compound has also been found to stimulate the immune system as described more fully below.

Description of the Prior Art In the search for effective drugs for the treat-ment of herpes viruses, considerable interest has been shown in the use of nucleoside analogues~ Previously, 5~iodo-2'-deoxyuridine (IUdR), cytosine arabinoside (Ara-C~, and arabinofuranosyladenine (Ara-A) have been found to be effective in varying degrees in the treatment of inEections due -to herpes viruses. EIowever, their hi.gh toxicity (espe-cially to rapi.dly proliferating cells), limits the useful-ness of these nucleoside analogues in -the treatment of viral infections.
.~ Recently 5-methoxymethyldeoxyuridine (~MUdR) has been synthesized and some inhibitory activity observed in monolayer cultures of secondary bovine fetal kidney cells against the cy-topathoqenic effects of bovine rhinotracheitis virus. This ~MUdR compound is a white powder of m.p.
J.20-125C having the formu:l.a:

~ CH2OC 3 3~ O `~' ~ .

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Summary o _ the Invention Animal tests have now shown -that MMUdR is active in vivo against certain herpes viruses, particularly herpes simplex (HSV-l). For example, significan-t activity in vivo has been demonstrated against herpes keratitis and herpes encephalitis. Animal tests have shown a synergistic activity against HSV-l induced keratitis infection, when MMUdR was used in conjunction with arabinosyl adenine (Ara-A). No significant toxicity was observed even at high doses and MMUdR appears to be devoid of teratogenic activity.
Animal tests also revealed an immunostimulant activity for MMUdR particularly at doses above about 500 mg/kg body weight.
The invention is directed to a pharmaceu-tical com-position for at least one of (a) trea-ting infections o-f herpes simplex viruses and (b) stimulating the immune system, in animals, comprising a physiologically-acceptable carrier sys-tem suitable for administration and 5-methoxymethyldeoxyuridine in an amoun-t effective to accomplish the desired effec-t. The compositions of the invention can include the 5-methoxymethyl-deoxyuridine in admixture with arabinosyl adenine. A pre-ferable composition comprises (a) 5-methoxymethyldeoxyuridine and (b) arabinosyl adenine, the relative amounts of (a) being from about 50 to about 200% wt. based on (b).
The compositions may be in appropriate dosage unit forms. Ihe compositions may be in the form of ar-tificial tear solutions for treatment of eye infections. The composi-tions can also include a local anaesthetic.
A much lower antiviral activity for MMUdR was observed against the following viruses in cell cultures:
equine rhinopneumonitis viruses, murine cytomegalovirus, feline rhinopneumonitis virus and vaccinia virus.

07~699 De.scrip-tion o_ Dca~in~s In t:he accompanying drc.wings Yigure l is a yraph of -the cumulative weighted lesion score vs. time in days after inoculating with virus (infec~ion) for different treating agents, with treatment commencincJ four hours post infection.
Figure 2 is similar to Figure l with increased concentration of treating agents.
Figure 3 is ~imilar to Elgure 2 with the treatment commencing 24 hours af-ter infection.

Det~iled Description and Preferred Embodiments The ~UdR compound can be administered parenterally or topically for therapeutic use, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, etc. The

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compourld may be dissolved or disp~rs~d in ~ny phy~ioloc~Lcally-accep-table llq~lid carrier, e.g., physiological saline, or ln 3ny suitable ointm~n-t base for topical application. M~UdR is water-soluble, and aqueous solutions of from about 1 to about 10% con-cen-tration are conveniently used. For -treatment of Herpes Keratitis (eye infection), a solution of 2-5% singlely and 1-2 in combination with 1-2% Ara-A has been found to be very effec-tive. The dosage of MMUdR can be any effective (antiviral or immunos-timulant) amount up to about 2000 mg/kg of body weight or more. A dose of 4000 mg/kg is the maximum dose administered and no mortality was observed in test animals after administration of this single dose. No morbidity or loss in weight was observed in animals even after administration of massive cumulative amounts of 15 g/kg total dosage. A preferred parenteral dosage range for treatment of systemic viral infections is from about 500 to about 2000 mg/kg. A preferred parenteral dosage range for immunostimu-lant purposes is from about 500 to about 2000 mg/kg. These do-! sages can be repeated at suitable intervals, e.g., daily, or more often if desired, until the desired effect is attained.
An enhanced ac-tivity against herpes simplex - virus has been observed when MMUdR is administered in conjunction with arabinosyl adenine (Ara-A). When this combined treatment is used, the relative proportions rnay vary widely but are usually within about 50 to about 200%
b~,~ weigh-t of MMUdR based on the Ara-A. The preferred antiviral dosage range for the combination is s:imilar -to that given above for MMUdR alone.
The Ara-A (9-~-D-arabinoEuranosyl adenine) has the for.-nula: Nl~2 ,fM
N ~ `N /

~1~)7'~699 This compound can also be administered by the same routes and in the same carriers as for the MMUdR. I-t is preferred to administer a mixture of the two compounds.
The following Examples are illustrative.
Example 1 TREATMENT OF ~IERPES SIMPLEX (HSV-1) KERATITIS
Comparative efficacy of MMUdR, IUdR and Ara-A in the treatmen-t of HSV-l induced keratitis in rabbit eye has been de-termined. Rabbits weighing 2~3 kg-were used in -these experiments. The eyes of each rabbit were anesthetized by instilling a couple of drops of ophthalmic proparicane (local anaesthetic 0.5% solution). The cornea was scarified (using 26 gauge needle) by making three ver-tical and three horizontal scratches on -the surface of the cornea. The degree and uniformity of scarification in each case was checked under u.v. light after fluorescein staining. One eye (left) of each rabbit was infected with 0.1 ml (1000 PFU plaque-forming units) of HSV-l by instilling the virus solution. The right eye was used as a toxicity control (to determine the effect of -the drug on corneal wound healing).
Treatment was initiated four hours after infection. Eye drops (1-2) were placed in bo-th eyes hourly or every two hours during the day (8 A M. - 6 P.M.) for 10 days. MMUdR, was dissolved in 1% polyvinyl alcohol and -tried a-t different concentrations (0.1 - 10%) and compared with oph-thalmic solu-tion of IUdR (Herplex (-trademarlc) 0.1%) and Ara-A suspension (5%). MMUdR and Ara-A were effec-tive at concen-trations -`2%
in contrast to IUdR which was effective a-t 0.1% Total days of treatment required were 8 -to 10 days for complete eradication of infection. These resul-ts indica-te tha-t MMUdR
was as potent as Ara-A. No corneal toxicity was observed wi-th MMUdR at concen-trations up to 10%.

~07~6~9 Thus, MMUdR is effec-tive in the trea-tment of herpes keratitis in animal tests. IUdR (0.1%) ophthalmic solution has been shown to he "teratogenic" in rabbits.
Therefore, even though MMUdR is less active than IUdR, because it is essentially devoid of mammalian toxicity, its use may be preferable in the treatment of herpes keratitis infections in humans.
Example 2 TREATMENT OF HERPES SIMPLEX (HSV-l) KERATITIS
Test Solut_n .
(i) MMUdR was dissolved in sterile physiological saline (0.85%). This solution was added to Adsorbotear (Trademark for an artificial tear solution), to give a final concen-tration of 20 mg/ml (2%) or 50 mg/ml (5%).
(ii) Ara-A (arabinosyl adenine) was suspended in Adsorbotear to give a final concentration of 20 mg/ml (2%) or 50 mg/ml (5%). In this vehicle Ara-A remains fairly suspended. How-ever, prior to use, the container was shaken to ensure that a homogeneous suspension was used in eye.
(iii) IUdR (Herplex, 0.1%) was used as supplied.
The comparative efficacy of MMUdR, IUdR, Ara-A
and combination of MMUdR + Ara-A in the treatment of HSV-l induced keratitis (deep stromal lesions) in rabbit eye was determined in further tests. Treatment was initiated ei-ther four or twen~ four hours after infection. On alternate days, each eye was examined, bo-th grossly and after fluores-cein staining for grading the severity of ocular lesions.
The method of Crown et al, (investigative ophthal, 2, 578 (1963)) as modified by Sidwell et al (Antimicro. Agents and Chemo. 3, 242 (1973)) for grading severity of ocular lesions, was used. The parameters measured were: infectivity (lesion size and type, corneal opacity) and Draize response (erythema, chemosis, discharge) on a weighted grading scale. (i) in-fectivity reading: scores of O (uninfected) to 4 (maximal severity); daily accumulation (infectivity scores x 10);
(ii) Draize parameters: similar scores - O (uninfected) -to 4 (maximal severity). The combined weighted infectivity and Draize scores of the control and drug-trea-ted eyes were then plotted together and compared. The results of these experiments (i.e. cumulative weighted score and days post virus inoculation) are summarized in Figures 1-3.
CONCLUSIONS:
(i) MMUdR at 2% and 5% concentration was found to have potent anti-keratitis activity in rabbit eye. On weight basis,it appears to be as potent as Ara-A.
(ii) MMUdR in combination with Ara-A showed synergistic effec-t against HSV-1-induced keratitis infection.
(iii) Antiherpes activity was seen by all scoring para-meters tested.
(iv) MMUdR was effective in inhibiting the inflammatcry (Draize) reac*ion to HSV-l virus.
(v) It was also effective in preventing the development of virus-induced lesions as determined by scoring of opacity and by microscopic examination of fluores-cein stained corneas.
(vi) No corneal toxicity was observed with up -to 10% con-centration of MMUdR.
Example 3 TREATMENT OF HERPES NCEPHALITIS (HSV-l) INFECTIONS
IN MI~E
Swiss mice (15 g) were inoculated intracerebrally with 50 pfu of HSV-l virus (-this virus dose has been found to produce 80-90% mortali-ty between 5-th and 10th days post infection). A single dose of MMUdR (in saline solution) was )7465~9 admil-~.is-tered a'L 500, 1,000, 1,500 ancl 2,0C0 m~/Xg/day, i.p.
fvr 5 days. Trea~inent was initiated 6 hours post inoculation of virus. Antiviral activity was assessed: (i) by increase in rnean survival time and (ii) survivors among the treated infected mice. The animals were observed for 21 days, and deaths occurring were recorded daily. The results are sur.~marized in Table I.

TABLE I

Efficacy of M~IUdR in the Treatment of Herpes Simplex Virusa Enchephali-tis-Induced Deaths in Mice.
.
Treatment Virus MMUdR MMUdR

CGntrols (1500 mg/Kg)b (2000 mg/Kg) ~ _ _ Survivors 3/10 7/10 9/10 Mean Survival Time 238 395 455c a50 pfu/mouse - intracerebrally; 15 gm Swiss mice.

bintraperitoneal daily for 5 days, treatment was initiated 6 hours post virus inoculation.

Cstatistically significant P <0.05.

CONCLUSIONS:
.
(:i) MMUdR at a dose of 1500 mg/Kg gave marked increase in life span (355 hr.) as compared to control mice (238 hr.) and also increased number of survivors.
(ii) MMUdR a-t 2,000 mg/Kg/day gave complete protection of ~ISV-l infected mice. One dea-th observed was due to secondary inEectiorl.

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~xample ~ I;Vi~'U~E R:E~SPONSE STUDIES ( 1`1 VI~'O) To assess the effects of MMUdR on immune respon-siveness, three an-tigens were studied - L-ISV-l, BruceLla abortus (Xilled) and sheep erythrocytes (SRBC). Groups of (BAI.B/C x C3H) Fl mice 8-10 weeks of age weighing be--tween 20-21 g were injected twice daily with varying doses (20-2,000 mg/Kg) of MMUdR The first daily injection was given intravenously (i.v.) and the second 8 hours later, intraperitoneally (i.p.). Drug treatments were continued for 9 days. To assess the effects of drug administration on immune responsiveness, 4 days after daily treatmerrt, animals were injected with one of the following antigens ~
0.2 rnl of 25% washed SRBC given i.v.; 10 killed Brucella abor-tus organisms given i.p. or 108 plaque forming units (pfu) equl~alen-ts of ultraviolet irradiated HSV~l given simultaneously. Mice receiving HSV-l antigen had been primed 6 weeks before by infection with HSV-l. Drug treat-men-ts were continued for 5 days after antigen administrations.
The results of these experiments are summarized in Tables II and III. Xt is apparent that immunosuppression was not induced even at the highest levels oE drug administration tested (2,000 mg/Kg). On the contrary, in both experiments, the numbers of antibody forming cells (~FC~ were higher in the groups that received the highest levels of drug than in the control group (Table II).
Drug administration also had no immunosuppressive effects against antibody responses to ~ISV-l or to BrucelLa ab_^t-us (Table III). Xn addition, -the clata included in r~ahle II also clearly sho~s -tha-t MMUdR was we:ll -tolerated by mice at :Least for the 9 clay treatrnent periocl.

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TABLE III

MEAN LOG2 ANTIBoDy TITERS OF _ICE IMMUNIZED WIT~
HEP~PES SIMPLEX TYP_ I AND BRUCEI,LA ABORTUS
Group Mean + SD Antibody Titer HSV-l Brucella Drug trea-ted 4.~5+0.4 4.5+0.7 Controls 4.5+0.8 5.0~

_ _ _ _ _ _ _ _ lAnimals were treated with 2000 mg/kg daily (divided into 2 equal doses) for 4 days, immunized with 10 killed _ cella abortus organisms and with 108 pfu equlvalents of UV-inactivated HSV-l. These mice had been infected with ~ISV-l 6 weeks previously. Drug administration was continued ~or a total of 9 days and serums were collec-ted a-t 10 days after immunization for antibody titra-tion~.
CONCLUSIONS:
(i) MMUd~ railed to show immunosuppressive activity up to doses as high as 2,000 mg/Kg given daily for 9 days.
(ii) There was evidence of stimulation of immune response after administration of MMUdR at the doses i.v., 1,000 mg/Kg; i.p., 2,000 mg/Kg.
EFFECT_F MMUdR ON IMMUNE RESPONSE (IN VITRO) Recovery from herpes virus infection possibly depends upon an intac-t lymphoid sys-tem. Therefore~ tes-ts were undertaken -to study the ef~'ects of MMUdR on lymphoid ~unctions. MMUdR did not have any immunosuppresive e-f'~ects on lymphoid fu'nctions. In contrast, in two preliminary trials, it was f`ound -to have immunostimulant ac-tivity.
This property is highly desirable for eradication of virus infections. MMUdR should be useful in counteracting the immunosuppressive properties of o-ther drugs.

Example 5 TEl~TOG~NIC ST-~JD~
_ _ _ _ _ ~ _ _ FiF~een bred female mice (Swiss) were injec-ted intraperitvneally once daily with 500 mg/Kg of ~MUdX.
Treatment was commenced on the day of initiation of breeding (as evidenced by sperm in vagina) and continued throughout gestation period (21 days). Altogether, appro-ximately 10 gm/Kg oE MMUdR was received by each pregnant female mouse. Control mice were given an equivalent amount of saline.
N rug e~fects were observed The young of the dosed and control groups showed comparable livability and body weight gains during the five-week nursing period. At weaning, physical examination and necropsy revealed no drug-related morphological abnor-malities.
Effect on the mouse fetus:

. .
On day 19 after initiation of treatment; fetuses from six treated (MMUdR, 500 mg/Kg, i.p. for 18 days) and six control (saline, i.p. for 18 d.ays) mice were removed .0 by caesarian and checked for pathology of reproductive tract of dam, resorption sites, litter size, welght, sex-ratio and any gross abnormalities. No di:Eferences were apparent between control and treated groups.

CON(~LUSION:
M~UclR appears to be devoid of teratogeni.c acti~ity in mice.

Sl~nary of Anima:L Experimerlts _._ _____ _ UdP~ was eY~tremely well tolerated aFter intraperi-toneal (i.p.) adrni.nist.ra-tion in micc-.

LDo - - 4000 mg/Kg (maxilnuln single close ac1min:lstered) I,Do = 2000 mg/KcJ/day for 10 days .

~ ~079~6~9 (ii) C].i.nical Chemistry ancl hemogram data of trea ed anir.als were normal.
(iii) MMUdR was found to have potent anti-keratitis activity in the rabbit eye.
(iv) In combination with Ara-A, MMUdR showed synergistic activity against ~SV-l induced kera-titis infection.
(v) MMUdR was effective in providing protection against ~SV-l induced encephalitis infection in mice.
(vi) MMUdR appeared to be devoid of tera-togenic activity in mice even after administration of massive doses of drug (500 mg/Kg/day, i.p.) for the entire gesta-tion period (20 days~.
(vii) MMUdR failed to elicit immunosuppressant activity in mice. Animals treated with doses as high as 2000 mg/Kg/day for 9 days produced normal immune responses to sheep erythrocy-tes, Brucella bacteria and Herpes Simp].ex virus. Lymphoid functions appeared normal.
(viii) M~IUdR showed stimulation of immune response at the higher doses.

Ophthalmic ointments of MMUdR can be prepared using white petrolatum mineral oil base (optionally containing an-hydrous lanolin) or polyethylene-gelled mineral oil base.
Preferably, concentrations in the range of about 5 to 15%
for r~UdR are used. Principal advantages of these prepara--tions are: (i) pro:Longed con-tact and effect, (ii) lack of irri-tation on initial instillation, and (ili) greater storage stability.

Claims (12)

1. A pharmaceutical composition for at least one of (a) treating infections of herpes simplex viruses and (b) stimulating the immune system, in animals, comprising a physiologically-acceptable carrier system suitable for administration and 5-methoxymethyldeoxyuridine in an amount effective to accomplish the desired effect.

2. The composition of claim 1 wherein carrier system is adapted for parenteral administration at a dosage within about 2 to about 3000 mg/kg body weight.

3. The composition of claim 1 wherein the compound is dispersed or dissolved in an aqueous liquid system.

4. The composition of claim 1 adapted for topical application in dispersion, solution or ointment form.

5. The composition of claim 1 including the 5-methoxy-methyldeoxyuridine in admixture with arabinosyl adenine.

6. The composition of claim 5 wherein the proportions of the two compounds are approximately equal.

7. The composition of claims 1, 3 or 4 in dosage unit form.

8. The pharmaceutical composition of claim 5 compri-sing (a) 5-methoxymethyldeoxyuridine and (b) arabinosyl adenine, the relative amounts of (a) being from about 50 to about 200% wt. based on (b).

9. The composition of claim 5, 6 or 8 in a dosage unit form.

10. The composition of claims 1, 5 or 8 including a physiological saline-base liquid carrier, or an ointment base.

11. The composition of claims 1, 5 or 8 in an artifi-cial tear solution.

12 . The composition of claims 1, 5 or 8 including a local anaesthetic.